Arrestins were first described as negative regulators of G protein-coupled receptor (GPCR) signaling via G proteins. New data show that the free and receptor-bound arrestins initiate signaling through MAP kinases, which regulate cell death, survival, and proliferation. In particular, both free and receptor- associated arrestin-3 scaffolds ASK1-MKK-JNK cascade, promoting JNK activation. Here we propose to elucidate the structural basis of arrestin-dependent activation of pro-apoptotic JNK family kinases and their activators MKK4/7 using biochemical and biophysical methods. The molecular mechanisms of the assembly of multi-protein signaling complexes (signalosomes) organized by arrestin-3 will be established, and arrestin-3 residues critical for JNK activation will be identified. Based on ths info, arrestin-3 mutants that bind ASK1, MKK and JNK, but do not promote JNK activation will be constructed. The potential of arrestin mutants with dramatically reduced ability to activate JNKs, several of which we already have, to protect cells against insults and prolong their survival will be tested. We have also constructed arrestin-3 mutants that activate JNKs more efficiently than parental wild type arrestin-3. We established the paradigm where arrestin-3-dependent JNK activation plays key role in cell survival. We will test the ability of hyperactive arrestin-3 mutants to facilitate cell death. We showed that inactive mutants tie up JNKs and upstream kinases in unproductive complexes, thereby acting in dominant- negative manner. We will test the potential of these mutants to protect cells and prolong their survival. Molecular toos that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases). PUBLIC HEALTH RELEVANCE: This proposal focuses on the elucidation of the structural basis of arrestin-dependent activation of pro- apoptotic JNK family kinases and their activators MKK4/7 using biochemical and biophysical methods. The potential of arrestin mutants with dramatically reduced ability to activate JNKs, that were constructed based on this info, to protect cells against insults and prolong their survival will be tested, as well as the ability of the mutats that activate JNKs more efficiently than wild type arrestins to facilitate cell death will be also tested. Molecular tools that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases).